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ALKBH7, a mitochondrial member of AlkB family, binds histones in vitro, part 2

ABSTRACT: Human genome encodes 9 AlkB family members, one of which is ALKBH7. In this study, we found recombinant ALKBH7 could bind bulk histones in GST pull-down and attenuate the signal of H4K20me2 in demethylation assays. With mass spectrometry, we also identified several potential ALKBH7-interacting proteins, such as NDUFS2, MRPS7 etc.; and found the expression of NDUFA4 and COX6C was down-regulated upon knockdown of ALKBH7. With these results, we can obtain a novel sight into the function of this protein.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Wei Ge

LAB HEAD: Wei Ge

PROVIDER: PXD008319 | Pride | 2020-05-26

REPOSITORIES: Pride

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			Action	DRS
	Humancanonical2015-07-02.fasta	Fasta
	MS150601-1-2.raw	Raw
	MS150601-10-2.raw	Raw
	MS150601-11-2.raw	Raw
	MS150601-12-2.raw	Raw

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Publications

The interactome and proteomic responses of ALKBH7 in cell lines by in-depth proteomics analysis.

Meng Shu S Zhan Shaohua S Dou Wanchen W Ge Wei W

Proteome science 20191229

<h4>Background</h4>ALKBH7 is a mitochondrial protein, involved in programmed necrosis, fatty acid metabolism, cell cycle regulation, and prostate cancer disease. However, the exact roles of ALKBH7 and the underlying molecular mechanisms remain mysterious. Thus, investigations of the interactome and proteomic responses of ALKBH7 in cell lines using proteomics strategies are urgently required.<h4>Methods</h4>In the present study, we investigated the interactome of ALKBH7 in mitochondria through im ...[more]

PMID: 31889914

Similar Datasets

ALKBH7, a mitochondrial member of AlkB family, binds histones in vitro, part 1

Project description:Human genome encodes 9 AlkB family members, one of which is ALKBH7. In this study, we found recombinant ALKBH7 could bind bulk histones in GST pull-down and attenuate the signal of H4K20me2 in demethylation assays. With mass spectrometry, we also identified several potential ALKBH7-interacting proteins, such as NDUFS2, MRPS7 etc.; and found the expression of NDUFA4 and COX6C was down-regulated upon knockdown of ALKBH7. With these results, we can obtain a novel sight into the function of this protein.

2020-05-26 | PXD008318 | Pride

ALKBH7, a mitochondrial member of AlkB family, binds histones in vitro, part 3

2020-05-26 | PXD008320 | Pride

Dynamic regulation of the COP9 signalosome: Insights into the spatial localization and subunit versatility of the complex

Project description:HeLa cells were fracitonated into nuclear soluble, chromatin bound and cytosolic. Extracts were immunoprecipitated with a CSN3 antibody, then eluted with glycine. Proteins were digested, then enriched for phosphopeptides. Samples were analyzed on an LTQ-Orbitrap. Data was analyzed using the TRansOMics software for time alignment and feature detection. MS/MS spectra searched using Mascot.

2019-11-25 | PXD000277 | Pride

Transcription profiling of rat at pheochromocytoma cell line PC12 treated with trimethyltin at low and high doses

Project description:The rat pheochromocytoma cell line PC12 cells were cultured in complete DMEM till 80% confluence, then placed at 5000 cells per squared cm. Cells were then plated in 24-well plates for cell viability assay and in T75 flasks for RNA isolation. Medium was replaced with serum-free fresh medium for 12 hours prior to TMT treatment. Gene expression patterns were then analysed using Rat Expression Array 230A Experiment Overall Design: In this study we analize gene expression patterns in PC12 cells treated with Trimethyltin (TMT). We utilized control cells (untreated) and two different concentration (1 and 5) Experiment Overall Design: We used three biological replicates, for the three concentration tested, according to MIAME guidelines Experiment Overall Design: (total 9 chips were used in this study).

2009-04-29 | E-GEOD-5073 | biostudies-arrayexpress

Reduced expression of mitochondrial complex I subunit Ndufs2 does not impact healthspan in mice

Project description:Aging in mammals leads to reduction in genes encoding the 45-subunit mitochondrial electron transport chain (ETC) complex I. It has been hypothesized that normal aging and age-related diseases such as Parkinson’s disease are in part due to modest decrease in expression of mitochondrial complex I subunits. By contrast, diminishing expression of mitochondrial complex I genes in lower organisms increases lifespan. Furthermore, metformin, a putative complex I inhibitor increases healthspan in mice and humans. In the present study, we investigated whether loss of one allele of Ndufs2, the catalytic subunit of mitochondrial complex I, impacts healthspan and lifespan in mice. Our results indicate that Ndufs2 hemizygous mice (Ndufs2+/-) show no overt impairment in motor function, learning, tissue histology, organismal metabolism, or sensitivity to metformin in a C57B6/J background. However, there are detectable changes at the level of gene expression in individual cell types and tissues due to loss of one allele of Ndufs2. Our data indicate that modest decline in expression of mitochondrial complex I subunit Ndufs2 is not detrimental to healthspan.

2021-12-12 | GSE190689 | GEO

H4K20me2 SILAC Nucleosome Affinity Purification

Project description:Goal of this project was the identification of chromatin interacting proteins whose binding is differentially regulated by di-methylation of lysine 20 on histone H4 (H4K20me2). To achieve this unodified and H4K20me2-modified histone H4 were generated by native chemical ligation and assembled into recombinant di-nucleosomes. The di-nucleosomes were immobilized on streptavidin-coated beads via the biotinylated di-nucleosomal DNA and used for nucleosome affinity purifications to identify proteins regulated by H4K20me2 from SILAC-labelled HeLaS3 nuclear extracts (Arg10 and Lys8). SILAC affinity purifications were carried out in "forward" (heavy extract on modified nucleosome and light extract on unmodified nucleosome) and "reverse" (light extract on modified nucleosome and heavy extract on unmodified nucleosome) label-swap experiments and protein abundances were quantified by MaxQuant.

2019-02-27 | PXD009281 | Pride

Benchmarking the Orbitrap Tribrid Eclipse for Next Generation Multiplexed Proteomics

Project description:The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a ~2X throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.

2022-01-07 | PXD016766 | Pride

Expression Analysis of Trimethyltin Chlorides (TMT) Impact on Embryonic Development

Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control Embryos were exposed to 10uM TMT or control from 48hpf to 60 or 72 hpf. Three replicates were collected for each time point. 40 embryos were pooled to comprise a replicate.

2011-03-23 | E-GEOD-28131 | biostudies-arrayexpress

A human interactome in three quantitative dimensions organized by stoichiometries and abundances

Project description:The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries and measures cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins, and introduces a framework for quantitative network analysis.

2015-10-23 | PXD002815 | Pride

An optimized microRNA scaffold increases shRNA processing and target knockdown

Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels). Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing. Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon. Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.

2014-11-24 | E-GEOD-62187 | biostudies-arrayexpress

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